The N-terminus specifies the switch between transport modes of the human serotonin transporter [Protein Structure and Folding]

The serotonin transporter (SERT) and other monoamine transporters operate in either a forward transport mode, where the transporter undergoes a full transport cycle, or an exchange mode, where the transporter seesaws through half-cycles. Amphetamines trigger the exchange-mode leading to substrate efflux. This efflux was proposed to rely on the N-terminus, which was suggested to adopt different conformations in the inward-, outward-facing and amphetamine-bound states. This prediction was verified by tryptic digestion of SERT-expressing membranes: in the absence of Na+, the N-terminus was rapidly digested. Amphetamine conferred protection against cleavage suggesting a relay between the conformational states of the hydrophobic core and the N-terminus. We searched for a candidate segment, which supported the conformational switch, by serial truncation removing 22 (ΔN22), 32 (ΔN32) or 42 (ΔN42) N-terminal residues. This did not affect surface expression, inhibitor binding and substrate influx. However, amphetamine-induced efflux by SERT-ΔN32 or SERT-ΔN42 (but not by SERT-ΔN22) was markedly diminished. We examined the individual steps in the transport cycle by recording transporter-associated currents: The recovery rate of capacitive peak - but not of steady-state - currents was significantly lower for SERT-ΔN32 than that of wild-type SERT and SERT-ΔN22. Thus, the exchange mode of SERT-ΔN32 was selectively impaired. Our observations show that the N-terminus affords the switch between transport modes. The findings are consistent with a model, where the N-terminus acts as a lever to support amphetamine-induced efflux by SERT.

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